Interleukin-6 (IL-6), originally identified as a B cell differentiation factor (Hirano et al. 1985, Proc. Natl. Acad. Sci, USA, 82: 5490-4; EP 0257406), is a multifunction cytokine that has a wide range of biological activities in various target cells and regulates—amongst others—immune responses, acute phase reactions, hematopoiesis, bone metabolism, angiogenesis, and inflammation (Nishimoto et al. 2006, Nat. Olin. Pract. Rheumatol. 2: 619-626). The interaction of IL-6 with IL-6 receptor (IL-6R) (Yamasak) et al. 1988, Science 241: 825-8; EP 0325474), an 80-kDa ligand-binding chain (IL-6R α-chain, or CD126), results in the formation of the IL-6/IL-6R complex. This complex binds to the membrane protein gp130 (Taga et al. 1989, Cell 58: 573-81; EP 0411946), a 130-kDa non-ligand-binding signal-transducing chain (IL-6R β-chain, or CD130) on a target cell, which transmits various physiological actions of IL-6. In cells with sufficient membrane-bound IL-6R, IL-6 binds to these receptors, the IL-6/IL-6R complex induces homodimerization of the gp130 molecule, and a high-affinity functional receptor complex of IL-6, IL-6R, and gp130 is formed (Hibi et al. 1990, Cell 63: 1149-1157). In cells that do not express sufficient cell-surface IL-6R, IL-6 signal transduction starts with the binding of IL-6 to the free, soluble form of IL-6R (sIL-6R), which lacks the membrane and intracytoplasmic portion of the 80-kDa membrane-bound IL-6R molecule (Taga et al. 1989, Cell 58: 573-581; Hibi et al. 1990, Cell 63: 1149-1157). Thus, either membrane-bound or soluble IL-6R can mediate IL-6 signal into cells, as long as the cells express gp130. Considerable amounts of sIL-6R are observed in serum and body fluids (Usón et al. 1997, J. Rheumatol. 24: 2069-2075; Desgeorges et al. 1997, 24: 1510-1516), and sIL-6R may play physiologic roles as well as having a pathologic role in immune-inflammatory and malignant diseases (Rose-John et al. 2006, J. Leukocyte Biol. 80: 227-236). Processes mediated via sIL-6R are indicated as trans-signaling.
Deregulation of IL-6 production is implicated in the pathology of several autoimmune and chronic inflammatory proliferative disease processes (Ishihara and Hirano 2002, Biochim. Biophys. Acta 1592: 281-96). IL-6 overproduction and signaling (and in particular trans-signaling) are involved in various diseases and disorders, such as sepsis (Starnes et al. 1999, J. Immunol. 148: 1968) and various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia (Klein et al. 1991, Blood 78: 1198-204), lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer. Non-limiting examples of other diseases caused by excessive IL-6 production or signaling include bone resorption (osteoporosis) (Roodman et al. 1992, J. Bone Miner. Res. 7: 475-8; Jilka et al. 1992, Science 257: 88-91), cachexia (Strassman et al. 1992, J. Clin. Invest. 89: 1681-1684), psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie et al. 1994, Int. J. Immunopharmacol. 16: 391-6), inflammatory diseases and disorder such as rheumatoid arthritis (RA), systemic onset juvenile idiopathic arthritis (JIA), hypergammaglobulinemia (Grau et al. 1990, J. Exp. Med. 172: 1505-8); Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus (Campbell et al. 1991, J. Clin. Invest. 87: 739-742).
As a consequence, inhibitors of IL-6 induced signaling have attracted much attention in the past (Hirano et al. 1990, Immunol. Today 11: 443-9). Polypeptides specifically binding to IL-6 (Klein et al. 1991, Blood 78: 1198-204; EP 0312996), IL-6R (EP 0409607) or gp130 (Saito et al. 1993, J. Immunol. Methods 163: 217-223; EP 0572118) proved to exhibit an efficient inhibitory effect on IL-6 functioning. Different antibodies and antibody fragments directed against human IL-6, against human IL-6R and against human gp130 protein for the prevention and treatment of IL-6 related disorders have been described. Examples are tocilizumab (Woo et al. 2005, Arthritis Res. Ther. 7: 1281-8; Nishimoto et al. 2005, Blood 106: 2627-32; Ito et al. 2004, Gastroenterology 126: 989-96; Choy et al. 2002, Arthritis Rheum. 46: 3143-50), BE8 (Bataille et al. 1995, Blood 86: 685-91; Emilie et al. 1994, Blood 84: 2472-9; Beck et al. 1994, N. Engl. J. Med. 330: 602-5; Wendling et al. 1993, J. Rheumatol. 20: 259-62) and CNTO-328 of Centocor (2004, Journal of Clinical Oncology 22/14S: 2560; 2004, Journal of Clinical Oncology 22/145: 2608; 2004, Int. J. Cancer 111: 592-5). Another active principle known in the art for the prevention and treatment of IL-6 related disorders is an Fc fusion of soluble gp130 (Becker et al. 2004, Immunity 21: 491-501; Doganci et al. 2005, J. Clin. Invest. 115: 313-25; Nowell et al. 2003, J. Immunol. 171: 3202-9; Atreya et al. 2000, Nat. Med. 6: 583-8). Immunoglobulin single variable domains directed against IL-6R and polypeptides comprising the same have been described in WO 08/020,079. Improved immunoglobulin single variable domains directed against IL-6R, have been described in WO 2010/115998 (see e.g. SEQ ID NOs: 60-72 of WO 2010/115998).
Tocilizumab is a humanized anti-human IL-6R antibody engineered by grafting the complementarily determining regions of a mouse anti-human IL-6R antibody into human IgG1κ to create a human antibody with a human IL-6R binding site (Sato et al. 1993, Cancer Res. 53: 851-856). Tocilizumab binds to the IL-6 binding site of human IL-6R and competitively inhibits IL-6 signaling. A series of clinical studies have shown that inhibition of IL-6 signaling by tocilizumab is therapeutically effective in RA, JIA, Castleman disease, and Crohn's disease (Nishimoto et al. 2003, J. Rheumatol. 30: 1426-1435; Nishimoto et al. 2004, Arthritis Rheum. 50: 1761-1769; Yokota et al. 2004, Autoimmun. Rev. 3: 599-600; Nishimote et al. 2005, Blood 106: 2627-2632; Ito et al. 2004, Gastroenterology 126: 989-996). In all of these diseases, tocilizumab ameliorated inflammatory manifestations and normalized acute phase protein levels, including C-reactive protein (CRP). Studies have confirmed 8 mg/kg every 4 weeks as the optimal dose and 4 mg/kg as the starting dose for the treatment of RA, with favorable efficacy and acceptable safety profiles. Tocilizumab 8 mg/kg every 4 weeks produced a sustained, adequate blockade of IL-6 receptors and normalized acute-phase reactants, such as C-reactive protein.
It was noticed that both serum IL-6 and serum sIL-6R increased in patients when IL-6 signaling was inhibited by tocilizumab while the disease symptoms continued to be ameliorated. Data showed that IL-6 temporarily increased following administration of tocilizumab. The increase was most likely caused by IL-6R blockade inhibiting clearance of IL-6 from the blood. Subsequently, there was a trend for decreasing IL-6 peak levels during 24 weeks for tocilizumab 8 mg/kg, suggesting decreased IL-6 production with amelioration of the disease or inflammatory status.
Following multiple doses of tocilizumab 4 or 8 mg/kg every 4 weeks for 24 weeks, mean sIL-6R levels increased with increasing treatment duration and reached a plateau at approximately weeks 8-12. For the 4 mg/kg dose, sIL-6R levels increased slightly with treatment duration. Peak sIL-6R levels were achieved in the middle of the dosing interval (i.e., at weeks 2, 6 and 14). The highest mean sIL-6R levels for tocilizumab 4 mg/kg were 5.1-5.6-fold above baseline. For the 8 mg/kg dose, mean sIL-6R levels remained high and increased with treatment duration, with minor fluctuations within the dosing interval. The highest mean sIL-6R levels for tocilizumab 8 mg/kg were 10-14-fold above baseline. The sustained increase in sIL-6R levels observed for the 8 mg/kg dose suggests persistent binding of tocilizumab to sIL-6R. At the 4 mg/kg dose, the fluctuating levels of sIL-6R suggest that tocilizumab exposure was below that for consistent binding of tocilizumab to sIL-6R. The accumulation of the sIL-6R in serum with an increasing number of tocilizumab infusions suggests that the tocilizumab/sIL-6R complex has a slower clearance than sIL-6R (Levi et al. 2008, Ann, Rheum. Dis. 67 (Suppl. II): 192).
Mean CRP normalized by week 2 of treatment with tocilizumab 8 mg/kg every 4 weeks and remained below the upper limit of normal through to week 24. By contrast, the improvement with tocilizumab 4 mg/kg was less striking and CRP concentrations fluctuated during the dosing interval (Smolen et al, 2008, Lancet 371: 987-997). Higher tocilizumab AUC (area under the curve for serum tocilizumab concentration-time profile from week 0-24) was associated with a more persistent low CRP level with a normal range from pooled pivotal Phase III studies (Levi et al. 2008, Ann. Rheum. Dis. 67 (Suppl. II): 192). Tocilizumab normalized the CRP levels in patients with RA as long as free tocilizumab remained≥1 ug/ml (Nishimoto et al. 2008, Blood 112: 3959-3964).
It was shown that after tocilizumab administration, more than 95% of the sIL-6R molecules were bound as immune complex, as long as the free tocilizumab concentration remained≥1 μg/ml (Nishimoto et al. 2008, Blood 112: 3959-3964). The relationship of tocilizumab, sIL-6R and CRP following single-dose tocilizumab administration (10 mg/kg) in RA patients is further illustrated in FIG. 1 (Schmitt et al, 2010, Clin. Pharmacol. Ther. 89: 735-740). (Zhan and Peck, 2011, Expert Rev. Clin. Pharmacol, 4: 539-558)